A method of modulating intestinal dysbiosis with a combination of probiotics

Abstract

The application discloses a method for regulating intestinal flora imbalance by using composite probiotics, and belongs to the technical field of probiotic microecological regulation. The method comprises the following steps: constructing a composite probiotic system containing Bifidobacterium longum subsp. infantis M-63, Lactobacillus acidophilus NCFM and Bifidobacterium bifidum; preparing a composite carbon source matrix containing water-soluble dietary fiber and insoluble dietary fiber; obtaining an acclimated composite flora with a qualified flora synergy index through dynamic co-cultivation gradient acclimation; embedding the acclimated composite flora in a microcapsule carrier with enteric characteristics to form a targeted release preparation; determining an initial administration dose and administration frequency according to the number ratio of the genus Bifidobacterium to the family Enterobacteriaceae in a fecal sample of a subject; and adjusting the administration dose in real time according to the acetic acid concentration and pH value during continuous administration. The method can enhance the synergistic metabolic capacity and intestinal adaptability of the composite probiotics, realize individualized dynamic administration, and improve intestinal flora imbalance.

Description

Technical Field

[0001] This invention relates to the field of probiotic microecological regulation technology, specifically a method for regulating intestinal flora imbalance using compound probiotics. Background Technology

[0002] Intestinal flora imbalance manifests as a decrease in the number of beneficial bacteria such as Bifidobacteria and an increase in the number of opportunistic pathogens such as Enterobacteriaceae. The ratio of Bifidobacteria to Enterobacteriaceae in feces is often used as an important reference for reflecting changes in flora structure. Current regulatory methods mostly involve directly supplementing with single strains or simple mixtures of probiotics, combined with conventional carbon sources or single dietary fibers to form ordinary preparations, and administered at fixed doses and frequencies. However, probiotic strains have varying abilities to utilize carbon sources. Unadapted bacteria entering the gut struggle to simultaneously utilize complex dietary fibers, leading to substrate competition between strains and unstable metabolic acid production. An unreasonable ratio of water-soluble to insoluble components in the dietary fiber matrix, or a lack of gradient adaptation, prevents the flora from obtaining suitable substrates in different intestinal segments, resulting in insufficient sustained production of short-chain fatty acids. Conventional enteric coating methods have limited effectiveness in maintaining bacterial activity during processing and storage, and the targeted release site and rate do not match the metabolic needs of the flora. Fixed dosing regimens ignore the differences in the degree of gut microbiota imbalance among subjects and do not stratify initial doses and dosing frequencies based on the ratio of Bifidobacterium spp. to Enterobacteriaceae in feces. During continuous dosing, metabolic indicators reflecting microbial fermentation activity, such as acetic acid concentration and pH value, are not included in the dose adjustment criteria, resulting in the intervention intensity not being dynamically adjusted according to changes in microbial status. These deficiencies lead to weak synergistic effects of compound probiotic preparations on the gut microbiota, low degree of individualized dosing, and unstable regulatory effects among different subjects. Summary of the Invention

[0003] The purpose of this invention is to provide a method for regulating intestinal flora imbalance using compound probiotics. This method involves constructing a specific compound probiotic system and formulating a compound carbon source matrix containing water-soluble and insoluble dietary fiber. A domesticated compound flora with a synergistic index is obtained through dynamic co-culture gradient domestication. This culture is then encapsulated in microcapsules with enteric properties to form a targeted release formulation. The initial dosing regimen is determined based on the ratio of Bifidobacterium to Enterobacterium species in the subject's feces. During continuous dosing, the dosage is dynamically adjusted based on acetic acid concentration and pH value, thereby achieving individualized intervention and stable regulation of intestinal flora imbalance.

[0004] To achieve the above objectives, the present invention provides the following technical solution: The present invention provides a method for regulating intestinal flora imbalance using compound probiotics, comprising: constructing a compound probiotic system containing *Bifidobacterium longum* subsp. infantis M-63, *Lactobacillus acidophilus* NCFM, and *Bifidobacterium bifidum*; based on the compound probiotic system, formulating a compound carbon source matrix containing water-soluble dietary fiber and insoluble dietary fiber; subjecting the compound probiotic system and the compound carbon source matrix to gradient domestication using a dynamic co-culture strategy to obtain a domesticated compound flora with a synergistic index reaching a preset requirement; encapsulating the domesticated compound flora in a microcapsule carrier with enteric properties to form a targeted release formulation; determining the initial dosage and frequency of the targeted release formulation according to the ratio of the number of *Bifidobacterium* to *Enterobacterium* in the subject's fecal sample; and dynamically monitoring the acetic acid concentration and pH value in the subject's fecal sample during continuous administration, and adjusting the dosage of the targeted release formulation for each subsequent administration in real time based on the monitoring results. This complex probiotic system works synergistically with a complex carbon source matrix. After gradient acclimatization and enteric-coated targeted release, it can promote the colonization and metabolism of beneficial bacteria in the intestine. Combined with individualized drug administration and dynamic feedback regulation, it can restore the balance of intestinal flora.

[0005] Preferably, in constructing the compound probiotic system, *Bifidobacterium longum* subsp. infantis M-63, *Lactobacillus acidophilus* NCFM, and *Bifidobacterium bifidum* are activated and cultured separately to obtain their respective primary seed cultures; the primary seed cultures are mixed at a volume ratio of 1:0.6:0.8 and inoculated into a liquid proliferation medium containing galactooligosaccharides and tryptone, and cultured under anaerobic conditions at 37°C for 12 hours with shaking to obtain the compound probiotic seed culture; the total number of viable bacteria in the compound probiotic seed culture is determined, and the viable bacteria concentration is adjusted to 1×10⁻⁶. 9 CFU / mL. The above strain ratio and culture conditions help maintain the balanced proliferation of the three strains, improve the viable cell density of the complex microbial community, and enhance its subsequent synergistic colonization ability.

[0006] Preferably, when preparing the composite carbon source matrix, fructooligosaccharides, inulin, and resistant dextrin are selected as the water-soluble dietary fiber components, and microcrystalline cellulose is selected as the insoluble dietary fiber component. The fructooligosaccharides, inulin, resistant dextrin, and microcrystalline cellulose are mixed in a mass ratio of 2:1:3:4, dissolved in phosphate buffer, the pH is adjusted to 6.8, and the mixture is autoclaved at 115°C for 15 minutes and cooled to 37°C for later use. The total fiber concentration of the composite carbon source matrix is ​​20 g / L. The combination of water-soluble and insoluble dietary fiber can provide a phased carbon source for the composite probiotic system, promoting the continuous proliferation and metabolic acid production of the bacterial community in the intestinal environment.

[0007] Preferably, in the dynamic co-culture strategy, the compound probiotic system is inoculated at a 5% inoculation rate into an acclimatization medium containing a first concentration of the compound carbon source matrix, and cultured at 37°C under anaerobic conditions for 24 hours to obtain a primary acclimatization culture. The primary acclimatization culture is then transferred at a 5% inoculation rate to an acclimatization medium containing a second concentration of the compound carbon source matrix, and cultured at 37°C under anaerobic conditions for 24 hours to obtain a secondary acclimatization culture. The secondary acclimatization culture is then transferred at a 5% inoculation rate to an acclimatization medium containing a third concentration of the compound carbon source matrix, and cultured at 37°C under anaerobic conditions for 24 hours to obtain a tertiary acclimatization culture, i.e., the acclimatized compound microbial community. The first concentration is lower than the second concentration, and the second concentration is lower than the third concentration. More preferably, the first concentration is 40% of the total fiber concentration of the compound carbon source matrix, the second concentration is 70% of the total fiber concentration of the compound carbon source matrix, and the third concentration is 100% of the total fiber concentration of the compound carbon source matrix. Dynamic co-culture with gradually increasing carbon source concentrations can enhance the adaptability of the complex microbial community to a high-concentration dietary fiber environment, and improve the synergistic index and metabolic stability of the microbial community.

[0008] As one technical solution of the present invention, the preparation of the targeted release formulation includes: centrifuging and collecting the domesticated complex bacterial flora and washing it three times with physiological saline to obtain bacterial sludge; mixing the bacterial sludge with sodium alginate solution at a volume ratio of 1:2 to form a mixed suspension, wherein the concentration of sodium alginate solution is 1.5%; extruding the mixed suspension into calcium chloride solution through a microdroplet generator to form microcapsules, wherein the concentration of calcium chloride solution is 2%; solidifying the microcapsules in calcium chloride solution for 30 minutes, washing them sequentially with deionized water and physiological saline, and then coating them in chitosan solution for 15 minutes, wherein the concentration of chitosan solution is 0.4% and the pH is 5.5; and freeze-drying the coated microcapsules to obtain the targeted release formulation. Enteric-coated microcapsule carriers can reduce the damage of gastric acid and bile salts to live bacteria, enabling the domesticated complex bacterial flora to be released in the intestinal tract, increasing the number of live bacteria reaching the intestine and the colonization efficiency.

[0009] Preferably, when determining the initial dosage and frequency of administration, fecal samples are collected from the subject, and the copy numbers of the 16S rRNA genes of *Bifidobacterium* and *Enterobacteria* are detected by real-time quantitative PCR. The ratio of the *Bifidobacterium* gene copy number to the *Enterobacteria* gene copy number is calculated to obtain the *Bifidobacterium* / *Enterobacteria* ratio. The *Bifidobacterium* / *Enterobacteria* ratio is compared with a grading threshold. The grading threshold includes a first threshold and a second threshold, where the first threshold is less than the second threshold. When the ratio is lower than the first threshold, the initial dosage is high-dose, and the administration frequency is twice daily. When the ratio is between the first and second thresholds, the initial dosage is medium-dose, and the administration frequency is once daily. When the ratio is higher than the second threshold, the initial dosage is low-dose, and the administration frequency is once every two days. Individualized initial dosage setting based on the relative abundance of *Bifidobacterium* and *Enterobacteria* in fecal samples can match the dosage intensity with the degree of intestinal flora imbalance, avoiding insufficient dosage or excessive intervention.

[0010] Preferably, during continuous administration, stool samples are collected from the subject before each administration. The concentration of acetic acid in the stool sample is detected using gas chromatography, and the pH value of the stool sample is measured using a pH meter. An acetic acid / pH adjustment coefficient is determined based on the acetic acid concentration and the pH value. This acetic acid / pH adjustment coefficient is compared with a target coefficient range to determine the dose adjustment range of the targeted release formulation for this administration. When the acetic acid / pH adjustment coefficient is lower than the lower limit of the target coefficient range, the dose for this administration is increased by one adjustment unit from the initial dose. When the acetic acid / pH adjustment coefficient is within the target coefficient range, the dose for this administration remains at the initial dose. When the acetic acid / pH adjustment coefficient is higher than the upper limit of the target coefficient range, the dose for this administration is decreased by one adjustment unit from the initial dose. More preferably, the lower limit of the target coefficient range is 0.8, the upper limit of the target coefficient range is 1.2, and the adjustment unit is 10% of the initial dose. By dynamically monitoring acetic acid concentration and pH value and adjusting the dosage in real time, the acidic metabolic environment of the intestine can be maintained within a suitable range, promoting the colonization of beneficial bacteria and inhibiting the excessive growth of opportunistic pathogens.

[0011] Preferably, on days 7, 14, and 21 of continuous administration, stool samples are collected again from the subjects to determine the ratio of Bifidobacterium spp. to Enterobacteriaceae, obtaining an updated ratio. Based on the updated ratio, the dosage and frequency of the targeted-release formulation in subsequent dosing cycles are re-determined; the subsequent dosing cycles are from day 22 to day 42. This phased reassessment of gut microbiota structure allows for dynamic optimization of the dosing regimen as the gut microbiota imbalance improves, enhancing long-term regulatory efficacy and safety.

[0012] The technical effects and advantages provided by the present invention in the above technical solution are as follows:

[0013] The compound probiotic system includes *Bifidobacterium longum* subsp. infantis M-63, *Lactobacillus acidophilus* NCFM, and *Bifidobacterium bifidum*. The compound carbon source matrix consists of fructooligosaccharides, inulin, resistant dextrin, and microcrystalline cellulose in a mass ratio of 2:1:3:4. During gradient acclimatization using a dynamic co-culture strategy, the compound probiotic system was sequentially inoculated into acclimatization media with total fiber concentrations of 40%, 70%, and 100%. Each acclimatization stage involved a 5% inoculum transfer and anaerobic culture at 37°C for 24 hours. As the carbon source concentration increased, the bacterial community gradually adapted to the substrate change from easily fermentable water-soluble fiber to insoluble fiber. Different strains developed specialized utilization and metabolic complementarity of the carbon source components, resulting in an acclimatized compound bacterial community with a synergistic index meeting the preset requirements. Compared to conventional simple mixing of strains and ungraded carbon source culture, the gradient-acclimated complex microbiota exhibited faster initiation of simultaneous fermentation of mixed dietary fiber, more sustained production of short-chain fatty acids, and better retention of activity and colonization tolerance after passing through the gastrointestinal tract, thus demonstrating stronger carbon source adaptability and metabolic stability in the intestinal environment. The initial dosage and frequency of the targeted-release formulation were determined based on the ratio of Bifidobacterium to Enterobacterium species in the subjects' fecal samples. When this ratio was below a first threshold, a high dose level and twice-daily dosing were used; when it was between the first and second thresholds, a medium dose level and once-daily dosing were used; and when it was above the second threshold, a low dose level and once every two days were used, ensuring that the initial dosing intensity directly corresponded to the subject's original intestinal microbiota imbalance. During continuous dosing, the acetic acid concentration and pH value in the fecal sample were measured before each dose, and the acetic acid / pH adjustment coefficient was calculated. This adjustment coefficient was compared with the target coefficient range; if it was below the lower limit, the adjustment unit was increased; if it was above the upper limit, the adjustment unit was decreased; and if it was within the target range, the initial dose was maintained. This dynamic adjustment method incorporates both gut microbiota structure and fermentation metabolism indicators into dosage decisions, enabling adjustments to subsequent dosages based on real-time changes in the subject's gut microbiota status. This avoids insufficient supplementation when gut microbiota imbalance is severe or continued high-dose supplementation after gut microbiota recovery, reducing the metabolic burden associated with dose mismatch and making the gut microbiota regulation process more aligned with individual dynamic needs. Attached Figure Description

[0014] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in this invention. For those skilled in the art, other drawings can be obtained based on these drawings.

[0015] Figure 1 This is a flowchart illustrating the method of using compound probiotics to regulate intestinal flora imbalance.

[0016] Figure 2 This is a flowchart of the construction of a compound probiotic system and the preparation of a compound carbon source matrix;

[0017] Figure 3 This is a flowchart of the preparation process for enteric-coated microcapsules targeting the release of a complex microbial flora;

[0018] Figure 4 It is a gradient acclimatization curve of live bacteria concentration change in a compound probiotic system with different total fiber concentrations and compound carbon source matrices;

[0019] Figure 5 These are the particle size distribution curves of microcapsules using different coating processes;

[0020] Figure 6 The curves showing the relationship between the copy number of 16S rRNA genes of Bifidobacterium and Enterobacteriaceae and the intensity of real-time fluorescence signals are shown.

[0021] Figure 7 This is a curve showing the changes in acetic acid concentration and pH value in fecal samples from subjects during continuous drug administration. Detailed Implementation

[0022] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0023] See Figure 1This invention provides a method for regulating intestinal flora imbalance using compound probiotics, comprising: constructing a compound probiotic system containing *Bifidobacterium longum* subsp. infantis M-63, *Lactobacillus acidophilus* NCFM, and *Bifidobacterium bifidum*; based on the compound probiotic system, formulating a compound carbon source matrix containing water-soluble dietary fiber and insoluble dietary fiber; subjecting the compound probiotic system and the compound carbon source matrix to gradient domestication using a dynamic co-culture strategy to obtain a domesticated compound flora with a synergistic index reaching a preset requirement; encapsulating the domesticated compound flora in a microcapsule carrier with enteric properties to form a targeted release formulation; determining the initial dosage and frequency of the targeted release formulation according to the ratio of *Bifidobacterium* to *Enterobacterium* species in the subject's fecal sample; and dynamically monitoring the acetic acid concentration and pH value in the subject's fecal sample during continuous administration, and adjusting the dosage of the targeted release formulation for each subsequent administration in real time based on the monitoring results.

[0024] In specific implementation, please refer to Figure 2 The construction of the compound probiotic system includes: activating and culturing *Bifidobacterium longum* subsp. infantis M-63, *Lactobacillus acidophilus* NCFM, and *Bifidobacterium bifidum* separately to obtain primary seed cultures of *Bifidobacterium longum* subsp. infantis M-63, *Lactobacillus acidophilus* NCFM, and *Bifidobacterium bifidum*. During the activation culture, single colonies of *Bifidobacterium longum* subsp. infantis M-63, *Lactobacillus acidophilus* NCFM, and *Bifidobacterium bifidum* were picked and inoculated into their respective liquid culture media, and cultured at 37°C under anaerobic conditions until the logarithmic growth phase. The primary seed cultures of *Bifidobacterium longum* subsp. infantis M-63, *Lactobacillus acidophilus* NCFM, and *Bifidobacterium bifidum* were then mixed at a volume ratio of 1:0.6:0.8 to obtain a mixed seed culture. The mixed seed culture was inoculated into a liquid proliferation medium containing galacto-oligosaccharides and tryptone, and cultured under anaerobic conditions at 37°C with shaking for 12 hours to obtain the compound probiotic seed culture. The total number of viable bacteria in the compound probiotic seed culture was determined, and the viable bacteria concentration was adjusted to 1×10⁻⁶. 9 CFU / mL. The total viable count was determined using the plate count method. The viable concentration was adjusted by dilution with sterile physiological saline or by centrifugation followed by resuspending.

[0025] In some embodiments, the preparation of the composite carbon source matrix includes: selecting fructooligosaccharides, inulin, and resistant dextrin as water-soluble dietary fiber components, and selecting microcrystalline cellulose as insoluble dietary fiber components. Fructooligosaccharides, inulin, resistant dextrin, and microcrystalline cellulose are weighed and mixed in a mass ratio of 2:1:3:4 to obtain a mixed fiber component. The mixed fiber component is dissolved in phosphate buffer, stirred until uniformly dispersed, and the pH is adjusted to 6.8 with hydrochloric acid solution or sodium hydroxide solution. It is then autoclaved at 115°C for 15 minutes and cooled to 37°C for later use. The total fiber concentration of the composite carbon source matrix is ​​20 g / L, meaning that each liter of the composite carbon source matrix contains a total mass of 20 grams of fructooligosaccharides, inulin, resistant dextrin, and microcrystalline cellulose.

[0026] In practice, when the compound probiotic system and the compound carbon source matrix are subjected to gradient acclimatization using a dynamic co-culture strategy, the compound probiotic system is inoculated into the acclimatization medium containing the first concentration of the compound carbon source matrix at a 5% inoculation rate. The inoculation volume of the compound probiotic system is 5% of the total volume of the acclimatization medium, and the viable bacteria concentration of the compound probiotic system is adjusted to 1×10⁻⁶ before inoculation. 9 CFU / mL. The total fiber concentration of the composite carbon source matrix was 20 g / L. The first concentration was 40% of the total fiber concentration of the composite carbon source matrix, corresponding to a total fiber concentration of 8 g / L in the composite carbon source matrix. The acclimatization medium inoculated with the composite probiotic system was cultured at 37°C under anaerobic conditions for 24 hours to obtain the primary acclimatization culture.

[0027] In some embodiments, the primary acclimatization culture is transferred at an inoculum volume of 5% to an acclimatization medium containing a second concentration of the composite carbon source matrix. The inoculum volume of the primary acclimatization culture is 5% of the total volume of the fresh acclimatization medium. The second concentration is 70% of the total fiber concentration of the composite carbon source matrix, corresponding to a total fiber concentration of 14 g / L in the composite carbon source matrix. The acclimatization medium inoculated with the primary acclimatization culture is cultured at 37°C under anaerobic conditions for 24 hours to obtain the secondary acclimatization culture.

[0028] Optionally, the secondary acclimatization culture is transferred at an inoculation rate of 5% to an acclimatization medium containing a third concentration of the composite carbon source matrix. The inoculation volume of the secondary acclimatization culture is 5% of the total volume of the fresh acclimatization medium. The third concentration is 100% of the total fiber concentration of the composite carbon source matrix, corresponding to a total fiber concentration of 20 g / L in the composite carbon source matrix. The acclimatization medium inoculated with the secondary acclimatization culture is then cultured at 37°C under anaerobic conditions for 24 hours to obtain the tertiary acclimatization culture, which is the acclimatized composite microbial community.

[0029] It is understandable that the first concentration is lower than the second concentration, and the second concentration is lower than the third concentration. The total fiber concentration in the composite carbon source matrix is ​​gradually increased by increasing the first, second, and third concentrations to achieve gradient acclimatization of the composite probiotic system. In each transfer operation, the primary and secondary acclimatization cultures are inoculated into fresh acclimatization medium at a rate of 5%, with the concentration of the composite carbon source matrix in the fresh acclimatization medium increasing sequentially to 40%, 70%, and 100%. The temperature is maintained at 37℃ during the cultivation process, and the anaerobic conditions remain unchanged. After the synergistic index of the bacterial community reaches the preset requirements, the tertiary acclimatization culture is used as the acclimatized composite bacterial community.

[0030] See Figure 4 In the figure, the left side of the vertical axis represents the viable bacterial concentration in CFU / mL, expressed on a logarithmic scale; the right side of the vertical axis represents the total fiber concentration in the composite carbon source matrix in g / L; and the horizontal axis represents the culture time in hours. The curves in the figure show the trends in viable bacterial concentration of Bifidobacterium longum subsp. infantis M-63, Lactobacillus acidophilus NCFM, and Bifidobacterium bifidum, and the corresponding changes in total fiber concentration, respectively.

[0031] The figure shows a dynamic co-culture system of the compound probiotics, inoculated with different concentrations of compound carbon source substrate at a 5% inoculum. A gradient acclimatization strategy was adopted, with the total fiber concentration of the compound carbon source substrate gradually increasing from an initial 8 g / L (40%) to 14 g / L (70%), and finally reaching 20 g / L (100%). The fiber concentrations at the three stages are clearly shown by dashed diamond markers, with each stage lasting 24 hours.

[0032] In the first phase (0 to 24 hours, total fiber concentration 8 g / L), the concentrations of all three probiotic strains increased significantly, with Bifidobacterium longum subsp. infantis M-63 reaching approximately 1 × 10⁻⁶. 9 CFU / mL, Lactobacillus acidophilus NCFM and Bifidobacterium bifidum reached approximately 8 × 10⁻⁶ CFU / mL. 8 CFU / mL and 7×10 8 CFU / mL indicates rapid bacterial proliferation under low total fiber concentration conditions.

[0033] After entering the second stage (24 to 48 hours, with the total fiber concentration increased to 14 g / L), the concentration of live probiotics decreased at the initial transfer, but then rapidly grew again to a stable level similar to that in the first stage, indicating that the bacterial community adapted to the acclimatization medium with a higher fiber concentration.

[0034] In the third phase (48 to 72 hours, total fiber concentration of 20 g / L), a temporary decrease in the initial viable bacteria concentration also occurred. Subsequently, the viable bacteria concentrations of the three probiotics continued to rise and tended to stabilize, with the final viable bacteria concentration of Bifidobacterium longum subsp. infantis M-63 approaching 1 × 10⁻⁶. 9CFU / mL, Lactobacillus acidophilus NCFM and Bifidobacterium bifidum remained stable at approximately 8 × 10⁻⁶ CFU / mL. 8 CFU / mL and 7×10 8 CFU / mL level.

[0035] In specific implementation, please refer to Figure 3 To form a targeted release formulation, the domesticated microbial community was encapsulated in a microcapsule carrier with enteric properties. The domesticated microbial community was collected by centrifugation and washed three times with physiological saline to obtain a bacterial sludge. During centrifugation, the domesticated microbial community was placed in a centrifuge container and centrifuged at 6000 rpm for 10 minutes at 4°C, discarding the supernatant. Sterile physiological saline was added to the centrifuged precipitate, vortexed to mix, and centrifuged at 6000 rpm for 10 minutes at 4°C, discarding the supernatant. This process was repeated three times to obtain the bacterial sludge.

[0036] In some embodiments, during the preparation of the sodium alginate solution, sodium alginate is weighed, dissolved in deionized water to prepare a 1.5% sodium alginate solution, stirred until completely dissolved, sterilized, and then cooled to room temperature. The mycelial sludge and sodium alginate solution are mixed at a volume ratio of 1:2 to form a mixed suspension. Before mixing, the volume of the mycelial sludge is measured, and the mixture is stirred to ensure the mycelial sludge is evenly dispersed in the sodium alginate solution.

[0037] Optionally, in the preparation of the calcium chloride solution, calcium chloride is weighed and dissolved in deionized water to prepare a 2% calcium chloride solution. The mixed suspension is then extruded into the calcium chloride solution through a microdroplet generator to form microcapsules. The nozzle diameter of the microdroplet generator is controlled to be 0.5 mm, and the mixed suspension is dripped into the calcium chloride solution through the nozzle to form spherical microcapsules.

[0038] It is understandable that the microcapsules are solidified in a calcium chloride solution for 30 minutes, allowing the sodium alginate and calcium chloride to undergo a cross-linking reaction. After solidification, the microcapsules are washed sequentially with deionized water and physiological saline to remove any residual calcium chloride solution from the surface. The microcapsules are washed three times with deionized water and three times with physiological saline.

[0039] In the chitosan solution preparation process, chitosan was weighed and dissolved in an acetate buffer solution with a pH of 5.5 to prepare a 0.4% chitosan solution. The washed microcapsules were then coated in the chitosan solution for 15 minutes, where chitosan was adsorbed onto the surface of the microcapsules via electrostatic attraction. After coating, the microcapsules were removed.

[0040] In the freeze-drying process, the coated microcapsules are placed in a freeze dryer, pre-frozen at -50°C for 2 hours, then heated to -20°C for vacuum drying for 12 hours, and then heated to 20°C for vacuum drying for 6 hours to obtain the targeted release formulation.

[0041] See Figure 5 The figure shows the particle size distribution curves of targeted release microcapsules prepared by different processes in this embodiment. The horizontal axis represents the microcapsule diameter in micrometers, ranging from 400 to 600 micrometers; the vertical axis represents the percentage corresponding to that particle size, expressed as a percentage, ranging from 0% to 0.85%. The figure includes three curves representing different preparation steps: microcapsules after solidification with calcium chloride solution (blue solid line, marked with dots), microcapsules after freeze-drying (green dashed line, marked with triangles), and microcapsules after coating with chitosan solution (orange dashed line, marked with squares).

[0042] After solidification of calcium chloride solution, the particle size distribution of microcapsules showed a single peak, with the peak value at approximately 520 micrometers, corresponding to a maximum percentage of about 0.67%. The curve was relatively symmetrical, indicating that the microcapsules formed during the solidification process had good particle size uniformity.

[0043] After freeze-drying, the peak value of the microcapsule particle size curve shifted significantly to the left, reaching approximately 480 micrometers, with a peak percentage of about 0.74%. Furthermore, the peak value of the curve was sharper than that of the microcapsule after calcium chloride curing, indicating that the freeze-drying process reduced the overall particle size of the microcapsules and made their distribution more concentrated.

[0044] The peak particle size of the microcapsules coated with chitosan solution shifted to the right to about 540 micrometers, with a peak percentage of about 0.61%. The curve was relatively broad and maintained a high percentage in the range of 500 to 580 micrometers, indicating that the coating operation increased the microcapsule particle size and slightly expanded the distribution range.

[0045] In practice, stool samples were collected from subjects to determine the initial dosage and frequency of the targeted-release formulation based on the ratio of Bifidobacteria to Enterobacteriaceae in their stool samples. During stool sample collection, freshly excreted stool samples (2-5 grams) were collected using sterile stool collection tubes. The samples were stored at 2-8°C and delivered for testing within 2 hours of collection. Total DNA was extracted from the stool samples using a fecal genomic DNA extraction kit, following the kit's instructions.

[0046] In real-time quantitative PCR (qPCR) detection, total DNA from fecal samples was used as a template to detect the copy numbers of the 16S rRNA gene in *Bifidobacterium* and *Enterobacteria*. *Bifidobacterium*-specific primers and probes were used for detection, while *Enterobacteria*-specific primers and probes were used for detection. The qPCR reaction system consisted of: qPCR premix, *Bifidobacterium*-specific primers and probes (or *Enterobacteria*-specific primers and probes), total DNA template from the fecal sample, and deionized water. The qPCR reaction conditions were set as follows: 95°C pre-denaturation for 5 minutes; 95°C denaturation for 15 seconds; 60°C annealing and extension for 30 seconds, for 40 cycles. Fluorescence signals were acquired using a qPCR instrument, and the copy numbers of the 16S rRNA gene in *Bifidobacterium* and *Enterobacteria* were calculated based on a standard curve.

[0047] In some embodiments, the Bifidobacterium / Enterobacterium ratio is calculated according to the formula:

[0048]

[0049] in, This indicates the ratio of Bifidobacteria to Enterobacteriaceae; This indicates the copy number of Bifidobacterium 16S rRNA gene obtained by real-time quantitative PCR, in copies / mL. This indicates the copy number of Enterobacteriaceae 16S rRNA gene obtained by real-time quantitative PCR, expressed in copies / mL. and All are positive numbers. Greater than zero.

[0050] Optionally, the Bifidobacterium / Enterobacterium ratio can be compared with grading thresholds to determine the initial dose level and dosing frequency level. The grading thresholds include a first threshold and a second threshold, with the first threshold being less than the second threshold. Both the first and second thresholds are preset values, set according to a grading standard for the degree of gut microbiota imbalance in the subject.

[0051] When the Bifidobacterium / Enterobacterium ratio is below the first threshold, the initial dose is a high dose level, administered twice daily. When the Bifidobacterium / Enterobacterium ratio is between the first and second thresholds, the initial dose is a medium dose level, administered once daily. When the Bifidobacterium / Enterobacterium ratio is above the second threshold, the initial dose is a low dose level, administered once every two days. The single dose of the high-dose targeted-release formulation is higher than that of the medium-dose targeted-release formulation, and the single dose of the medium-dose targeted-release formulation is higher than that of the low-dose targeted-release formulation.

[0052] See Figure 6 The horizontal axis in the figure uses a logarithmic scale to represent the copy number of 16S rRNA genes in Bifidobacteria and Enterobacteriaceae, in units of copies / mL, ranging from 10² to 10. 8 The vertical axis represents the fluorescence signal intensity obtained by real-time quantitative PCR detection, with arbitrary units. The legend distinguishes two curves: one is the detection signal of Bifidobacterium-specific primers and probes (blue solid line with dots), and the other is the detection signal of Enterobacteriaceae-specific primers and probes (red dashed line with squares).

[0053] The curves generally showed a linear increase in fluorescence signal intensity with increasing gene copy number, indicating that the real-time quantitative PCR detection system has good sensitivity and quantification capabilities. The Bifidobacterium detection curve consistently appeared above the Enterobacteriaceae detection curve, suggesting that at the same gene copy number level, the fluorescence signal intensity of the Bifidobacterium primers and probes was slightly higher, indicating that the amplification efficiency of the Bifidobacterium primer-probe system was superior to that of the Enterobacteriaceae system.

[0054] The several markers on the curve are evenly distributed, ranging from 10², 10³, and 10... 4 10 5 10 6 10 7 Up to 10 8 The fluorescence signal intensity ranged from approximately 40 to over 110, indicating that the detection system exhibited good linear response over a wide dynamic range. The logarithmic scale on the horizontal axis and the linear scale on the vertical axis together reflect that this PCR detection method is suitable for quantifying microbial genes from low to high abundance, meeting the accurate detection requirements for Bifidobacterium and Enterobacteriaceae gene copy numbers in fecal samples in this embodiment.

[0055] In practice, during continuous drug administration, the concentration of acetic acid and pH value in the subject's stool samples are dynamically monitored, and the dosage of the targeted release agent for each subsequent administration is adjusted in real time based on the monitoring results. Stool samples are collected from the subject before each administration. The collection procedure uses sterile stool collection tubes to collect freshly excreted stool samples, with a collection volume of 2 to 5 grams. The stool samples are stored at 2°C to 8°C, and the acetic acid concentration and pH value are measured within one hour of collection.

[0056] Acetic acid concentration was detected using gas chromatography (GC). In the GC procedure, 1 gram of fecal sample was weighed, diluted with deionized water to 10 mL, vortexed for 5 minutes, and centrifuged at 12,000 rpm for 10 minutes at 4°C. The supernatant was filtered through a 0.45 μm filter to obtain the test solution. The test solution was injected into the GC-chromatograph equipped with a flame ionization detector. The chromatographic column was a DB-FFAP capillary column, 30 meters long, with an inner diameter of 0.25 mm and a membrane thickness of 0.25 μm. The GC conditions were set as follows: injection port temperature 200°C, detector temperature 250°C, column oven initial temperature 80°C, held for 1 minute, increased to 180°C at 10°C / min, and held for 2 minutes. A series of standard solutions were prepared using acetic acid standards. The peak areas of the standard solutions were measured under the same GC conditions to establish a standard curve of acetic acid concentration versus peak area. The concentration of acetic acid in the fecal sample was determined from the standard curve based on the peak area of ​​the test solution.

[0057] pH values ​​were measured using a pH meter. During the pH measurement procedure, 2 grams of fecal sample were weighed, 20 ml of deionized water was added, the mixture was vortexed until homogeneous, and allowed to stand at room temperature for 5 minutes. The pH meter electrode was then inserted into the supernatant, and the pH value was read. Before use, the pH meter was calibrated using three standard buffer solutions with pH values ​​of 4.00, 6.86, and 9.18.

[0058] In some embodiments, the acetic acid / pH adjustment coefficient is calculated based on the acetic acid concentration and pH value. The acetic acid / pH adjustment coefficient is calculated according to the formula:

[0059]

[0060] in, Indicates the acetic acid / pH adjustment coefficient; This indicates the concentration of acetic acid in a fecal sample obtained by gas chromatography, expressed in millimoles per liter. This indicates the pH value of a fecal sample obtained by measuring with a pH meter; it is a dimensionless value. It is a positive number. Greater than zero.

[0061] The acetic acid / pH adjustment coefficient was compared with the target coefficient range to determine the dose adjustment range of the targeted release formulation during this administration. The lower limit of the target coefficient range was 0.8, and the upper limit was 1.2. The lower limit of the target coefficient range of 0.8 and the upper limit of the target coefficient range of 1.2 were set based on the following: the mean acetic acid / pH adjustment coefficient of fecal samples from normal healthy individuals is 1.0. The lower limit of the target coefficient range was set at 0.8, and the upper limit of the target coefficient range was set at 1.2. When the acetic acid / pH adjustment coefficient is within the target coefficient range, it indicates that the balance between the acetic acid production level and the fecal pH in the subject's fecal sample is as expected.

[0062] When the acetic acid / pH adjustment coefficient is below the lower limit of the target range, the dose for this administration is increased by one adjustment unit from the initial dose. When the acetic acid / pH adjustment coefficient is within the target range, the dose for this administration remains the initial dose. When the acetic acid / pH adjustment coefficient is above the upper limit of the target range, the dose for this administration is decreased by one adjustment unit from the initial dose. The adjustment unit is 10% of the initial dose. The dose after increasing by one adjustment unit is the sum of the initial dose and the initial dose multiplied by 10%. The dose after decreasing by one adjustment unit is the difference between the initial dose and the initial dose multiplied by 10%.

[0063] Optionally, on days 7, 14, and 21 of continuous drug administration, stool samples were collected again from the subjects. The copy numbers of the 16S rRNA gene in *Bifidobacterium* and *Enterobacteria* were detected by real-time quantitative PCR, and the ratio of *Bifidobacterium* to *Enterobacteria* gene copy numbers was calculated to obtain an updated ratio. In the real-time quantitative PCR detection procedure, total DNA was extracted from the re-collected stool samples. Using the total DNA from the stool samples as a template, *Bifidobacterium*-specific primers and probes were used to detect the 16S rRNA gene copy number in *Bifidobacterium*, and *Enterobacteria*-specific primers and probes were used to detect the 16S rRNA gene copy number in *Enterobacteria*. The real-time quantitative PCR reaction conditions were set as follows: 95℃ pre-denaturation for 5 minutes; 95℃ denaturation for 15 seconds; 60℃ annealing and extension for 30 seconds, for 40 cycles. The copy numbers of 16S rRNA genes of Bifidobacterium and Enterobacteriaceae were calculated based on the standard curve. The ratio of the copy numbers of Bifidobacterium genes to Enterobacteriaceae genes was then calculated to obtain the updated ratio.

[0064] The updated ratio is compared with the grading thresholds to redetermine the dosage and frequency of the targeted-release formulation in subsequent dosing cycles. The grading thresholds include a first threshold and a second threshold, with the first threshold being less than the second threshold. When the updated ratio is below the first threshold, the subsequent dosage is at a high dose level, and the subsequent dosing frequency is twice daily. When the updated ratio is between the first and second thresholds, the subsequent dosage is at a medium dose level, and the subsequent dosing frequency is once daily. When the updated ratio is above the second threshold, the subsequent dosage is at a low dose level, and the subsequent dosing frequency is once every two days. The subsequent dosing cycle is from day 22 to day 42.

[0065] See Figure 7 In the graph, the horizontal axis represents the continuous administration time (days), the left vertical axis represents the acetic acid concentration (millomoles per liter), and the right vertical axis represents the pH value. The solid blue curve and its shaded area represent the acetic acid concentration and its fluctuation range, respectively, while the dashed red curve and its shaded area represent the pH value and its fluctuation range. Marked points every few days indicate specific detection time points on the curves.

[0066] As shown in the figure, the acetic acid concentration exhibited a significant upward trend with continuous administration, gradually increasing from approximately 10 mmol / L initially to approximately 22 mmol / L on day 20, nearly doubling overall. This indicates that the administration of the targeted-release formulation promoted the production of acetic acid in the subjects' intestines. Simultaneously, the pH value showed a slow downward trend, gradually decreasing from approximately 7.1 initially to around 6.8, and then stabilizing after a brief inflection point around day 14, indicating a slight acidification of the intestinal environment.

[0067] The increase in acetic acid concentration and the slight decrease in pH value correspond to each other, consistent with the technical solution in this embodiment, which involves dynamically monitoring the acetic acid concentration and pH value in the subject's feces, calculating the acetic acid / pH adjustment coefficient, and adjusting the dosage accordingly. The trend in the figure shows that with continuous administration of the targeted release formulation, the metabolic activity of the intestinal flora in the subject increases, the level of acetic acid production rises, and the intestinal pH tends to be within a suitable range, which is beneficial for regulating intestinal flora imbalance.

[0068] Furthermore, the pH value fluctuated only slightly, remaining stable between 6.7 and 7.1 without any abnormal fluctuations, indicating a good acid-base balance in the intestinal environment during drug administration. Changes in acetic acid concentration and pH value jointly affect the acetic acid / pH adjustment coefficient, and the trend in the figure reflects the effective functioning of the dynamic feedback mechanism described in this embodiment, which uses this coefficient to adjust the dosage in real time.

[0069] The above description is merely a specific embodiment of this application, but the scope of protection of this application is not limited thereto. Any changes or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in this application should be included within the scope of protection of this application.

Claims

1. A method for regulating intestinal flora imbalance using compound probiotics, characterized in that, include: Construct a complex probiotic system containing Bifidobacterium longum subsp. infantis M-63, Lactobacillus acidophilus NCFM, and Bifidobacterium bifidum; Based on the aforementioned compound probiotic system, a compound carbon source matrix containing water-soluble dietary fiber and insoluble dietary fiber was formulated. The compound probiotic system and the compound carbon source matrix are subjected to gradient domestication according to a dynamic co-culture strategy to obtain a domesticated compound microbial community with a microbial community synergy index that meets the preset requirements. The domesticated complex microbial community was encapsulated in a microcapsule carrier with enteric properties to form a targeted release formulation; The initial dosage and dosing frequency of the targeted release formulation were determined based on the ratio of the number of Bifidobacteria to Enterobacteriaceae in the subject's fecal sample. During continuous administration, the concentration of acetic acid and pH value in the subject's fecal samples are dynamically monitored, and the dose of the targeted release formulation is adjusted in real time for each subsequent administration based on the monitoring results.

2. The method for regulating intestinal flora imbalance using compound probiotics according to claim 1, characterized in that, In the step of constructing a compound probiotic system containing Bifidobacterium longum subsp. infantis M-63, Lactobacillus acidophilus NCFM, and Bifidobacterium bifidum: Bifidobacterium longum subsp. infantum M-63, Lactobacillus acidophilus NCFM and Bifidobacterium bifidum were activated and cultured separately to obtain their respective primary seed cultures; The primary seed culture was mixed at a volume ratio of 1:0.6:0.8 and inoculated into a liquid proliferation medium containing galactooligosaccharides and tryptone. The mixture was then cultured under anaerobic conditions at 37°C for 12 hours with shaking to obtain the compound probiotic seed culture. The total number of viable bacteria in the compound probiotic seed liquid was determined, and the viable bacteria concentration was adjusted to 1×10⁻⁶. 9 CFU / mL.

3. The method for regulating intestinal flora imbalance using compound probiotics according to claim 1, characterized in that, In the step of preparing a composite carbon source matrix containing water-soluble dietary fiber and insoluble dietary fiber: Fructooligosaccharides, inulin, and resistant dextrin were selected as water-soluble dietary fiber components, while microcrystalline cellulose was selected as insoluble dietary fiber components. The oligofructose, inulin, resistant dextrin and microcrystalline cellulose were mixed in a mass ratio of 2:1:3:4, dissolved in phosphate buffer, the pH was adjusted to 6.8, and the mixture was autoclaved at 115°C for 15 minutes and cooled to 37°C for later use. The total fiber concentration of the composite carbon source matrix is ​​20 g / L.

4. The method for regulating intestinal flora imbalance using compound probiotics according to claim 1, characterized in that, In the step of gradient domestication of the compound probiotic system and the compound carbon source matrix according to a dynamic co-culture strategy to obtain a domesticated compound microbial community with a synergistic index reaching a preset requirement: The compound probiotic system was inoculated at a rate of 5% into an acclimatization culture medium containing the first concentration of the compound carbon source matrix, and cultured at 37°C under anaerobic conditions for 24 hours to obtain a primary acclimatization culture. The primary acclimatization culture was transferred at an inoculum rate of 5% to an acclimatization medium containing the second concentration of the composite carbon source matrix, and cultured at 37°C under anaerobic conditions for 24 hours to obtain the secondary acclimatization culture. The secondary acclimatization culture was transferred at an inoculation rate of 5% to an acclimatization medium containing the third concentration of the composite carbon source matrix, and cultured at 37°C under anaerobic conditions for 24 hours to obtain the tertiary acclimatization culture, which is the acclimatization composite microbial community. The first concentration is lower than the second concentration, and the second concentration is lower than the third concentration.

5. The method for regulating intestinal flora imbalance using compound probiotics according to claim 4, characterized in that, The first concentration is 40% of the total fiber concentration of the composite carbon source matrix, the second concentration is 70% of the total fiber concentration of the composite carbon source matrix, and the third concentration is 100% of the total fiber concentration of the composite carbon source matrix.

6. The method for regulating intestinal flora imbalance with compound probiotics according to claim 1, characterized in that, In the step of encapsulating the domesticated complex microbial community in a microcapsule carrier with enteric properties to form a targeted release formulation: The domesticated complex microbial community was collected by centrifugation and washed three times with physiological saline to obtain microbial sludge; The bacterial sludge was mixed with sodium alginate solution at a volume ratio of 1:2 to form a mixed suspension, wherein the concentration of sodium alginate solution was 1.5%. The mixed suspension was extruded into a calcium chloride solution using a microdroplet generator to form microcapsules, wherein the concentration of the calcium chloride solution was 2%. After the microcapsules were solidified in calcium chloride solution for 30 minutes, they were washed with deionized water and physiological saline in sequence, and then coated in chitosan solution for 15 minutes. The concentration of chitosan solution was 0.4% and the pH was 5.

5. The coated microcapsules are freeze-dried to obtain the targeted release formulation.

7. The method for regulating intestinal flora imbalance with compound probiotics according to claim 1, characterized in that, In the step of determining the initial dosage and dosing frequency of the targeted release formulation based on the ratio of Bifidobacterium genus to Enterobacterium family in the subject's fecal sample: Fecal samples were collected from the subjects, and the copy number of 16S rRNA genes of Bifidobacterium and Enterobacteriaceae was detected by real-time quantitative PCR. The ratio of the copy number of the Bifidobacterium gene to the copy number of the Enterobacteriaceae gene is calculated to obtain the Bifidobacterium / Enterobacterium ratio. The Bifidobacterium / Enterobacterium ratio is compared with a grading threshold to determine the grade of the initial dosage and the grade of the dosing frequency; The grading threshold includes a first threshold and a second threshold, wherein the first threshold is less than the second threshold; When the ratio is lower than the first threshold, the initial dosage is at a high dose level, and the dosing frequency is twice daily. When the ratio is between the first threshold and the second threshold, the initial dosage is a medium dose and the dosing frequency is once daily. When the ratio is higher than the second threshold, the initial dosage is a low dose level, and the dosing frequency is once every two days.

8. The method for regulating intestinal flora imbalance with compound probiotics according to claim 1, characterized in that, In the step of dynamically monitoring the acetic acid concentration and pH value in the subject's fecal samples during continuous administration, and adjusting the dosage of the targeted release agent for each subsequent administration in real time based on the monitoring results: Fecal samples were collected from subjects before each administration. The concentration of acetic acid in the fecal samples was detected by gas chromatography, and the pH value of the fecal samples was measured by a pH meter. Based on the acetic acid concentration and the pH value, the acetic acid / pH adjustment coefficient is calculated, and the acetic acid / pH adjustment coefficient is equal to the quotient of the acetic acid concentration divided by the pH value; The acetic acid / pH adjustment coefficient is compared with the target coefficient range to determine the dose adjustment range of the targeted release formulation during this administration. When the acetic acid / pH adjustment coefficient is lower than the lower limit of the target coefficient range, the dose administered this time will be increased by one adjustment unit based on the initial dose; When the acetic acid / pH adjustment coefficient is within the target coefficient range, the dose administered this time shall be maintained at the initial dose. When the acetic acid / pH adjustment coefficient is higher than the upper limit of the target coefficient range, the dose administered this time will be reduced by one adjustment unit from the initial dose.

9. A method for regulating intestinal flora imbalance using compound probiotics according to claim 8, characterized in that, The lower limit of the target coefficient range is 0.8, and the upper limit of the target coefficient range is 1.

2. The adjustment unit is 10% of the initial dosage.

10. The method for regulating intestinal flora imbalance with compound probiotics according to claim 1, characterized in that, On days 7, 14, and 21 of continuous administration, stool samples were collected again from the subjects to determine the ratio of Bifidobacterium spp. to Enterobacteriaceae, and the updated ratio was obtained. Based on the updated ratio, the dosage and frequency of the targeted release formulation in subsequent dosing cycles are re-determined; The subsequent dosing period is from day 22 to day 42.

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